Correct orientation of the mitotic spindle during cell division is critically important in development as well as in the regulation and maintenance of stem cell populations in tissues. Orienting the spindle in the appropriate plane of division is an active process mediated by membrane-anchored proteins and subject to regulation by mitotic kinases. Molecular motors are thought to act as force generators that both orient and anchor the spindle in the proper plane of division. We have found that depletion of a little studied kinesin family motor, Kif25, in human cells causes misorientation of the mitotic spindle and defects in spindle assembly. We have determined that Kif25 is a C-terminal kinesin motor although the structure and function of this motor are poorly understood. The focus of the current proposal is to test the hypothesis that Kif25 regulates both spindle assembly and orientation by producing force on spindle microtubules from its centrosomal location. Single molecule studies will be used to characterize the microtubule-dependent activity of this novel motor. These experiments will compliment and inform our investigations of Kif25 in live cells where we will use a combination of live and fixed cell imaging to evaluate protein depletions and dominant negative constructs, as well as the identification and characterization of Kif25 interacting partners. Overal this proposal will fully characterize Kif25 function and contribute an important new component to the machinery controlling spindle orientation in mitotic cells.